Human amnion epithelial cell therapy reduces hypertension-induced vascular stiffening and cognitive impairment

Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. However, current anti-hypertensive drugs do not treat the underlying tissue damage, such as inflammation-associated fibrosis. Human amnion epithelial cells have several properties amenable for treating vascular pathology. This study tested the effect of amnion epithelial cells on vascular pathology and cognitive impairment during hypertension. Male C57Bl6 mice (8–12 weeks) were administered vehicle (saline; n = 58) or angiotensin II (0.7 mg/kg/d, n = 56) subcutaneously for 14 d. After surgery, a subset of mice were injected with 106 amnion epithelial cells intravenously. Angiotensin II infusion increased systolic blood pressure, aortic pulse wave velocity, accumulation of aortic leukocytes, and aortic mRNA expression of collagen subtypes compared to vehicle-infused mice (n = 9–11, P < 0.05). Administration of amnion epithelial cells attenuated these effects of angiotensin II (P < 0.05). Angiotensin II-induced cognitive impairment was prevented by amnion epithelial cell therapy (n = 7–9, P < 0.05). In the brain, amnion epithelial cells modulated some of the inflammatory genes that angiotensin II promoted differential expression of (n = 6, p-adjusted < 0.05). These findings suggest that amnion epithelial cells could be explored as a potential therapy to inhibit vascular pathology and cognitive impairment during hypertension.


Assessment of aortic stiffening
Ultrasound imaging of the abdominal aorta was performed on days 0 and 14 post-surgery using a Vevo 2100 (VisualSonics; FUJIFILM; Canada) 12 .Mice were anaesthetised using isoflurane (2% at 0.4 L/min) and placed on a heated platform.Abdominal fur was removed and ultrasound transmission gel (Aquasonics, USA) was applied to the abdomen.Pulse wave doppler images and EKV retrospective acquired B-Mode images were obtained from longitudinal sections of the abdominal aortas (suprarenal) using a MS-400 ultrasound transducer.Data were exported and pulse wave velocity was analysed using the VevoLab and VevoVasc software (FUJIFILM Visualsonics Inc. Canada) and InD-V loop method.

Flow cytometric analysis of leukocytes in the aorta
Flow cytometry was performed as previously described in brain tissue but modified for the aorta and blood 13 .Mice were killed by carbon dioxide asphyxiation and perfused via the left ventricle with 0.2% clexane (400 IU, Sanofi Aventis, Australia) in 0.01 M phosphate-buffered saline (PBS).The entire aorta (aortic arch to femoral bifurcation) with perivascular fat was harvested for flow cytometry.Samples were minced with scissors and digested in PBS (with MgCl 2 and CaCl 2 ) containing a mixture of collagenase type XI (125 U/ml), collagenase type I-S (460 U/ml) and hyaluronidase (60 U/ml) (Sigma-Aldrich, USA) for 30 min at 37 °C.Samples were then passed through a 70 μm filter.
Whole blood was collected via the right ventricle into heparinised tubes.Samples were then incubated with 10 mL of red blood cell (RBC) lysis buffer for 5 min at room temperature on a shaker.Samples were washed with PBS and centrifuged at 4 °C for 5 min (1500 RPM).This was repeated to ensure that erythrocytes were removed from the sample.Cell were then resuspended with 500 μL PBS.

Gene expression in the aorta
Messenger RNA expression of collagen in the aorta was determined using TaqMan ® real-time PCR.The aorta was harvested and snap frozen in liquid nitrogen.Aortae were sonicated in TRIzol™ (Life Technologies, USA), mixed with chloroform, and centrifuged at 824 × g for 15 min at 4 °C.The aqueous phase was collected and RNA was extracted using the RNeasy ® Micro Kit (Qiagen, USA).RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and converted to 1 st strand cDNA using High Capacity cDNA RT Kit (Applied Biosystems, USA).Commercially available primers (Applied Biosystems, USA) were used to measure mRNA expression of collagen (Col1a1, Col3a1, Col4a1, Col5a1), and a house-keeping gene, Gapdh, on a CFX96 Touch Real-Time PCR Detection machine (Bio-Rad, USA).Changes in gene expression were assessed using the delta-delta C T method 14 .

Immunolocalisation of amnion epithelial cells
Amnion epithelial cells were localised in the aorta using immunohistochemistry.Fixed (4% paraformaldehyde), paraffin-embedded thoracic aorta sections (5 μm) were dewaxed, incubated with histolene (2 × 10 min), rinsed with 100% and 70% ethanol and then distilled H 2 O. Antigen retrieval was performed using citrate buffer (pH 6.0), sections were then washed with PBS and endogenous peroxidase was blocked with 1% H 2 O 2 .Endogenous mouse IgG was blocked using goat anti-mouse IgG followed by blocking with 10% donkey serum in phosphate buffered saline.Sections were then incubated overnight at 4 °C with anti HLA-G (1:500; Ab52455; Abcam, UK).The next day, sections were washed and incubated with a horse radish peroxidase-conjugated donkey anti-mouse secondary antibody (1:200) for 45 min at room temperature.Sections were washed with PBS and incubated in DAB brown solution.Following this, sections were washed, counterstained with haemotoxylin and mounted with DPX mounting media.Images were captured with an Olympus DP73 Camera (Olympus Corporation, Tokyo, Japan) connected to an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) at 400 × magnification running CellSens Standard Software (version 1.17, Olympus Corporation).

Picrosirius red staining
For picrosirius red staining, aortae were prepared and sectioned as described above.Five μm sections were incubated with 0.3% Picrosirius red solution (PolysciencesInc.,USA) for 1.5 h at room temperature.Sections were washed with acidified water, rinsed with 100% ethanol and histolene before moutning with DPX mounting media.Sections were imaged using a polarized microscope (Olympus BX53, Japan) and analysed for percentage collagen content by ImageJ.Two aortic sections per mouse were analysed by an investigator blinded to the treatment groups.

Behavioural testing
We used the open field test to evaluate locomotor activity and anxiety-like behavior 15,16 , and working memory using the novel object recognition test 17 .Mice were acclimatized to the testing apparatus by placing them in the empty box for 10 min per day for 2 days prior to testing.The open field test was performed on day 2 of acclimation.A 30 × 30 cm zone was set up in the middle of the box (inner zone) and a 10 cm wide zone around the edges of the box (outer zone).Time in the inner zone as well as total distance travelled was tracked using Ethovision XT (Noldus Information Technology BV, Wageningen, The Netherlands).On the day of testing, the mouse was placed in the box containing two identical objects and allowed to explore for 10 min.One hour later, the mouse was placed back in the same box for 5 min with one of the familiar objects replaced by a novel object.Interactions with the objects (defined as nose entering a 2 cm zone around the object) were tracked using Ethovision XT.Mice than did not interact with the objects for > 10 s in total were excluded from the analysis.

Brain RNA sequencing
RNA sequencing was performed as previously described 18 .In brief, brain hemispheres were harvested and snap frozen in liquid nitrogen.Brains were sonicated in TRIzol™ (Life Technologies, USA), mixed with chloroform, and centrifuged at 824 × g for 15 min at 4 °C.The aqueous phase was collected and RNA was extracted using the RNeasy ® Micro Kit (Qiagen, USA).RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and then stored at − 80 °C.The RNA samples were sent to NovogeneAIT Genomics (Singapore) for cDNA library preparation and RNA sequencing.mRNA was purified from total RNA using poly-T oligoattached magnetics.mRNA was converted to cDNA and purified using AMPure XP Beads (Beckman Coulter Life Sciences, USA).cDNA libraries were acquired by PCR amplification.High-throughput sequencing was conducted using the HiSeqTM2500 platform (Illumina, USA).The results were mapped to the Ensembl-released mouse genome sequence and annotation.Differential expression analysis was conducted using the DESeq R Package V.1.10.1 and P-values were adjusted using the Banjamini and Hochberg's approach for controlling the false discovery rate.Genes were considered differentially expressed if the adjusted P-value was less than 0.05.R package heatmap3 and log2Fold-Change output from EdgeR V.3.2.4 were used to create heatmaps for differentially expressed genes.

Statistical analysis
Results are expressed as mean ± S.E.M. Sample size calculations were performed with G Power Software (version 3.1.5).Normality of the data was checked using a Shapiro-Wilk test and was normally distributed.Statistical analyses between groups were performed using Student's unpaired t-test, one-sample t-test, or a one-or two-way ANOVA followed by a Tukey's or Sidak's post-hoc test, as appropriate.P < 0.05 was considered to be significant.RNA sequencing data were analysed using R Studio.GraphPad Prism software (version 9.0, GraphPad Software Inc., USA) was used to perform all other statistical analyses.

Amnion epithelial cells infiltrate the aorta in hypertensive mice
To localise amnion epithelial cells in the aorta, we stained sections with human leukocyte antigen G (HLA-G) 19 .As expected, no HLA-G positive staining was observed in angiotensin II-infused mice that received saline injection (Fig. 1A).By contrast, HLA-G positive cells (arrow in higher magnification view) were observed in the  www.nature.com/scientificreports/adventitia of angiotensin II-infused mice that had received amnion epithelial cells 14 d previously (Fig. 1A).No positive staining was observed in medial or intimal layers of the aorta.

Amnion epithelial cells reduce systolic blood pressure and aortic pulse wave velocity
Infusion of angiotensin II for two weeks resulted in an elevation in systolic blood pressure of ~ 60 mmHg (Fig. 1B).Administration of amnion epithelial cells blunted the pressor response to angiotensin II by 20 mmHg (Fig. 1B).Treatment with amnion epithelial cells did not affect systolic blood pressure in vehicle-infused mice (Fig. 1B).Aortic pulse wave velocity measured by ultrasound sonography was lower in angiotensin II-infused mice treated with amnion epithelial cells (Fig. 1C,D).

Amnion epithelial cells reduce aortic expression of collagen genes
Angiotensin II infusion increased mRNA expression of Col1a1, Col3a1 and Col5a1 (Fig. 3A, B and D) but not Col4a1 (Fig. 3C).These changes in gene expression were prevented by administration of amnion epithelial cells (Fig. 3A, B and D).Aortic collagen deposition was also assessed using Picrosirius red staining (Supplementary Fig. 2).We did not observe any significant changes in collagen 1, collagen 3 or the collagen 1:collagen 3 ratio.

Amnion epithelial cells improve working memory in hypertensive mice
Neither the % time spent in the inner zone (Fig. 4A) or total distance travelled (Fig. 4B) was affected by hypertension or amnion epithelial cell administration.Intact working memory was defined as spending significantly more than 50% of the interaction time exploring the novel object.Representative heat maps for each treatment group with the location of novel and familiar objects are shown in Fig. 4C.Vehicle-infused mice spent more time (~ 60%) interacting with the novel object than the familiar object (Fig. 4D; one sample t-test vs 50%), whereas angiotensin II-infused mice did not discriminate between novel and familiar objects (Fig. 4D).Amnion epithelial cell therapy did not adversely impact the performance of vehicle-infused mice (Fig. 4D).Moreover, administration of amnion epithelial cells in angiotensin II-infused mice resulted in more time spent interacting with the novel object than the familiar object (~ 70%; Fig. 4B).

Amnion epithelial cells modulate expression of genes in the brain
Bulk RNA sequencing was performed on the brains of mice infused with vehicle, angiotensin II, vehicle + amnion epithelial cells or angiotensin II + amnion epithelial cells.A heatmap showing all differentially expressed genes is shown in Supplementary Fig. 3.When comparing mice infused with angiotensin II alone versus vehicle alone, there were 341 differentially expressed genes (168 upregulated and 173 downregulated) in the brain (Fig. 5A).There were 365 differentially expressed genes (183 upregulated and 182 downregulated) in the brain when comparing mice infused with angiotensin II alone versus angiotensin II + amnion epithelial cells (Fig. 5B).

Discussion
The major finding of this study is that amnion epithelial cells can prevent aortic stiffening, inflammation and cognitive impairment induced by hypertension.Specifically, amnion epithelial cells limited the pressor response to angiotensin II, prevented angiotensin II-induced aortic immune cell infiltration and expression of collagen, prevented impairment of working memory and modulated angiotensin II-induced transcriptomic changes in the brain.Our findings are consistent with some of the known anti-inflammatory and anti-fibrotic properties of amnion epithelial cells.Thus, amnion epithelial cells may be a potential therapeutic option for the prevention or treatment of hypertension-induced vascular injury and cognitive impairment.Amnion epithelial cells blunted the pressor response to angiotensin II by 20 mmHg.Previously, amnion epithelial cells have been found to prevent pulmonary hypertension in experimental lung injury 20 .To our knowledge, this study is the first to examine the effects of amnion epithelial cells in a model of systemic hypertension.The blood pressure lowering effect of amnion epithelial cells could at least partly be due to their immunomodulatory properties.There is now a large body of evidence supporting a role of the immune system in hypertension 21 , with immune cells such as T cells 22 and macrophages 23 having been demonstrated to contribute to hypertension.In this study, angiotensin II promoted infiltration of leukocytes (specifically macrophages and inflammatory monocytes) into the aorta, and this could be prevented by co-adminstration of amnion epithelial cells.We have previously shown that amnion epithelial cells home to sites of acute injury (i.e.stroke 7 or traumatic brain injury 19 ).In the present study we identified amnion epithelial cells in the aorta, which we and others have shown to be a site of inflammation and injury during hypertension.As hypertension causes systemic injury, it is also possible that amnion epithelial cells migrate to other tissues.We have also shown that a CCR2 antagonist reduces blood pressure and aortic macrophage numbers in mice infused with angiotensin II 24 .Similarly, depletion of monocytes and macrophages via treatment with clodronate 23 or low-dose diptheria toxin 25 attenuated the pressor response to angiotensin II and this was restored by adoptive transfer of monocytes 25 .Furthermore, we have reported these cells to have anti-inflammatory properties in animal models of lung injury 26,27 and stroke 7 .In this study, amnion epithelial cells reduced macrophage and monocyte infiltration in the aorta.Amnion epithelial cells can inhibit macrophage migration in vitro through production of macrophage migration-inhibitory factor (MIF) 28 .We previously demonstrated that amnion epithelial cells can reduce macrophage infiltration in a mouse model of lung injury, and decrease chemotaxis of macrophages towards recombinant mouse macrophage inflammatory protein 2 in vitro 29 .Hence, it is possible that amnion epithelial cells in this study reduced aortic macrophage infiltration at least in part by inhibiting their migration.Amnion epithelial cells do not appear to affect macrophage proliferation or survival 28,29 .Thus, the anti-hypertensive effect of amnion epithelial cells may, in part, be due to their ability to suppress innate immune cell infiltration into the aorta.We and others have shown that amnion epithelial cells can reduce fibrosis in mouse models of liver fibrosis 8,30,31 and hepatic stellate cells co-cultured with amnion epithelial cells have reduced collagen production 8,32 .Vascular stiffening results from structural changes such as excessive fibrosis (including increased collagen) and reduced elastin.Angiotensin II infusion increased both expression of aortic collagen genes and aortic pulse wave velocity in vivo 33 , consistent with the development of aortic stiffening 12 .By contrast, administration of amnion epithelial cells reduced expression of collagen and pulse wave velocity in angiotensin II-infused mice, consistent with antifibrotic actions that limited vascular stiffness in hypertension.
Angiotensin II caused cognitive impairment (impaired working memory assessed using the novel object recognition test), but did not impair locomotor activity or anxiety-like behaviour, and this was prevented by amnion epithelial cells.This protection of working memory could have occurred at least in part by reducing vascular stiffening.Vascular stiffening is a predictor of cognitive decline 34 and increases in aortic pulse wave velocity are associated with impaired memory and executive function 4,35 .Increased pulse wave velocity reflects elevated pulsatile pressure in the brain which can damage the cerebral vasculature 36 and lead to breakdown of the blood brain barrier (BBB), neuroinflammation, neurodegeneration and cognitive decline 37 .It is also possible that the BBB breakdown that occurs with hypertension 38 may enable amnion epithelial cells to enter the brain parenchyma and protect against hypertension-induced cognitive impairment.Other anti-fibrotic therapies have also been reported to improve cognition.Interestingly, idiopathic pulmonary fibrosis is associated with aortic stiffening 39 and cognitive deficits 40 .Furthermore, pirfenidone, an anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis, can prevent scopolamine-induced cognitive impairment 41 , improve neurological outcome in a rat model of traumatic brain injury 42 , and inhibit angiotensin II-induced cardiac hypertrophy and fibrosis 43 .A limitation of the novel object recognition test is that it cannot distinguish between degrees of impairment.Thus, we can only conclude that angiotensin II treatment impaired working memory and that amnion epithelial cells prevented this.To determine degrees of impairment, a different cognitive test (e.g.Barnes maze) would need to be performed.Perivascular macrophages have been shown to contribute to hypertension-induced cognitive impairment 44 .The effect of amnion epithelial cells on perivascular macrophages is not currently known, however, amnion epithelial cells have been reported to reduce microglial activation in the brain in a rat model of stroke 45 .Microglia are the resident macrophages of the brain and microglia activation can contribute to cognitive decline in a

Figure 3 .
Figure 3. Administration of amnion epithelial cells prevents angiotensin II-induced aortic expression of collagen.The effect of angiotensin infusion and co-treatment with amnion epithelial cells (AECs) on mRNA expression of (A) collagen type 1 alpha 1 chain, (B) collagen type 3 alpha 1 chain, (C) collagen type 4 alpha 1 chain and (D) collagen type 5 alpha 1 chain in the aorta (n = 6-8).All data are mean ± S.E.M. *P < 0.05.Two-way ANOVA with Sidak's test.

Figure 4 .
Figure 4. Administration of amnion epithelial cells improves angiotensin II-induced cognitive impairment.(A) Percentage of time spent in the inner zone in the open field test.(B) Total distance travelled (in cm) in the open field test (C) Representative heatmap plots showing interaction between familiar and novel objects in mice infused with vehicle, angiotensin II, vehicle + amnion epithelilal cells (AECs) and angiotensin II + AECs.(D) Effect of angiotensin II infusion and co-treatment of AECs on recognition memory (n = 7-9).All data are mean ± S.E.M. *P < 0.05.One-sample t-test versus 50%.

Figure 5 .
Figure 5. Administration of amnion epithelial cells modulates genes that are differentially expressed in the brain by angiotensin II-infusion.Volcano plot of differentially expressed genes in brains of mice infused with (A) vehicle versus angiotensin II or (B) angiotensin II versus angiotensin II + AECs.The threshold of differential expression is p-value < 0.05.The horizontal axis is the log2 fold change of genes.The vertical axis is statistical significance scaled as − log 10 p-value.Each dot represents an individual gene (blue: no significant difference; red: upregulated expression; green: downregulated expression).The top upregulated and downregulated genes in brains of mice infused with (C) vehicle versus angiotensin II or (D) angiotensin II versus angiotensin II + AECs (n = 6 per group).Upregulated genes in red and downregulated genes in blue.The colour scale represents the log10 (average FPKM + 1) value.